Molecular and developmental approach to understanding diversification in mediophyte diatoms

硅藻（微小的海洋植物）具有重要的生态和经济意义；事实上，每吸入五分之一的氧气来自硅藻，但有些会产生对人体有害的神经毒素。它们也是种类最丰富（约20万种）的微藻群，因此可以与陆地上开花植物的多样性相媲美。从进化的角度来看，和开花植物一样，硅藻是相对较新的物种，这表明它们的物种形成率非常高。我的长期研究目标是评估与硅藻繁殖相关的过程和结构的进化意义，以了解自侏罗纪时期化石记录中硅藻首次出现以来，各种性别类型在硅藻壮观的多样化中可能发挥的作用。本提案的直接目标是将生殖、发育和分子方法应用于硅藻进化史上两个主要转变的代表，以理清它们彼此之间以及与它们最近的亲属之间的关系。我建议研究两个硅藻目，这两个目目前在传统和分子系统发育（“家谱”）上似乎都不太适合或不确定。类海硅藻与它们最近的亲戚有很大的不同，而类海硅藻很可能拥有证据表明，它们是最先进的硅藻——pennate的近亲。我们将寻找这些不合适或不确定的特征是如何形成的发展指标。我们将使用先进的显微技术（光和荧光显微镜，扫描电子显微镜，x射线微分析，二氧化硅特异性染色）来检测和定位乳白色二氧化硅（硅藻细胞壁的组成部分），并描述其发育阶段细胞壁成分的精细结构。我们还将使用迄今未充分利用的分子工具，ITS2共识二级结构模型，在我们的系统发育推断中。我们的侦察工作表明，这些模型在解开硅藻系统发育关系方面与在绿藻和其他植物中一样有用。这些硅藻的系统发育分析与我们在这里提出的个体发生方法相结合是硅藻进化生物学的新方法。我们在大约十年前率先采用了这种综合方法，并且是世界上唯一一个将生殖数据系统地添加到硅藻进化生物学更广泛的实验室。进一步发展的主要障碍是缺乏可重复的性别诱导协议，我们现在已经为所涉及的硅藻群开发了这种协议。这项工作的结果将提供一个多管齐下的框架，以更好地了解硅藻的主要进化转变。拟议的工作包括许多新的培训机会，包括微生物培养，高级显微镜，以及为各级初级合作者提供的分子和生物信息学项目。

Diatoms (microscopic marine plants) are ecologically and economically important; indeed, the oxygen in every fifth inhalation originates from diatoms, but some produce neurotoxins harmful to humans. They are also the most species-rich (~200,000) group of microalgae, thus rivaling the diversity of flowering plants on land. Evolutionarily, and like flowering plants, diatoms are relatively new arrivals, suggesting an exceptionally high speciation rate. My long-term research objective is to evaluate the evolutionary significance of the processes and structures associated with diatom reproduction in order to understand what role various types of sexuality might have played in the spectacular diversification of diatoms since their first appearance in the Jurassic Period fossil record. The immediate objectives specific to this proposal are to apply reproductive, developmental and molecular approaches to representatives of two major transitions in diatom evolutionary history to untangle their relationships to each other and to their closest relatives. I propose to investigate two orders of diatoms that currently seem to poorly or uncertainly fit in both traditional and molecular phylogenies ("family trees"). Thalassiosiroids differ dramatically from their closest relatives, and biddulphioids are likely to hold evidence that indicates the closest living relative of the most advanced of the diatoms, the pennates. We will search for developmental indicators of how these ill-fitting or uncertain characters came to be. We will use advanced microscopical techniques (light and epifluorescence microscopy, scanning electron microscopy, X-ray microanalysis, silica-specific stains) to detect and localize opaline silica (the building blocks of diatom cell walls) and describe the fine structure of the cell-wall components during their developmental stages. We will also use a heretofore underutilized molecular tool, ITS2 consensus secondary structure models, in our phylogenetic inferences. Our reconnaissance work has shown that these models are as useful in untangling diatom phylogenetic relationships as they have proven to be in green algae and other plants. Phylogenetic analysis of these diatoms combined with the ontogenetic approach we propose here is novel in diatom evolutionary biology. We pioneered this integrative approach only about a decade ago and are the only laboratory in the world to systematically add reproductive data to the wider body of diatom evolutionary biology. The major obstacle to further advancement is the lack of reproducible sex-induction protocols, which we now have developed for the diatom groups involved. The results of this work will provide a multi-pronged framework to better our understanding of major evolutionary transitions in diatoms. The proposed work includes many new training opportunities, including microbial culturing, advanced microscopy, and molecular and bioinformatics projects for junior collaborators at all levels.